Post-transcriptional regulation of messenger RNA (mRNA) stability and translation is important for controlling gene expression. This project seeks to generate and utilize structural information to enhance our understanding of these processes. RNA silencing, the destruction of mRNA by double stranded RNA containing corresponding sequences, has proven to be a powerful tool to knock out expression of target genes in eukaryotic cells and has therapeutic potential. In the past few years, much has been learned about the mechanism by which RNA silencing occurs, including the identification of proteins involved in the process. PUF proteins are regulators of gene expression important for embryonic development and germline stem cell maintenance. This project has two main focuses. The first is to study proteins that suppress RNA silencing or are involved in the RNA silencing pathway. We are using structural and biochemical methods to understand their functions. We have been studying the RNA recognition properties of a viral protein that inhibits RNA interference by binding to small interfering RNAs. Our studies indicate that this protein is capable of binding to small RNAs with mismatched and bulged bases at many positions, suggesting that the protein will inhibit many microRNA pathways by binding to duplex precursor RNAs. We have also determined the solution structure of the double-stranded RNA binding domain from the enzyme Drosha, which processes primary microRNAs to pre-microRNAs (ref. 3). This structure reveals that the domain retains RNA-binding features and suggests that the domain may be involved in substrate recognition. The second focus is to study PUF family proteins. We have determined the crystal structure of the PUF protein yeast Puf4p alone and in complex with RNA (ref. 1). These structures identify adaptations of this protein to recognize RNA target sequences with an additional base relative to a prototypical PUF protein human Pumilio1. We have also determined crystal structures of yeast Puf3p and C. elegans fem-3 binding factor (FBF) in complex with their RNA targets. These structures reveal recognition elements that expand our understanding of RNA recognition and post-transcriptional gene regulation by the PUF family of proteins. By comparing recognition properties of these proteins and other PUF proteins in their respective organisms, we can better understand how specific RNA targets are regulated by particular PUF proteins (manuscripts submitted). Our studies on the RNA recognition properties of PUF proteins have allowed us to create artificial splicing factors in collaboration with Dr. Zefeng Wang's lab at the Univ. of North Carolina. Together we have demonstrated the ability to design a factor that can regulate alternative splicing of endogenous pre-mRNA in cells (ref. 4).